In-vivo evaluation of the response of Galleria mellonella larvae to novel copper(II) phenanthroline-phenazine complexes

Herein we report the in-vivo characterisation and metabolic changes in Galleria mellonella larvae to a series of bis-chelate copper(II) phenanthroline-phenazine cationic complexes of [Cu(phen) ₂ ] ²⁺ (Cu-Phen), [Cu(DPQ)(Phen)] ²⁺ (Cu-DPQ-Phen) and [Cu(DPPZ)(Phen)] ²⁺ (Cu-DPPZ-Phen) (where phen = 1,10-phenanthroline, DPQ = dipyrido[3,2-ƒ:2′,3′-h]quinoxaline and DPPZ = dipyrido[3,2-a:2′,3′-c]phenazine). Our aim was to investigate the influence of the systematic extension of the ligated phenazine ligand in the G. mellonella model as a first step towards assessing the in-vivo tolerance and mode of action of the complex series with respect to the well-studied oxidative chemical nuclease, Cu-Phen. The Lethal Dose ₅₀ (LD ₅₀ ) values were established over dose ranges of 2 – 30 μg at 4-, 24-, 48- and 72 h by mortality assessment, with Cu-Phen eliciting the highest mortality at 4 h (Cu-Phen, 12.62 μg < Cu-DPQ-Phen, 21.53 μg < Cu-DPPZ-Phen, 26.07 μg). At other timepoints, a similar profile was observed as the phenazine π-backbone within the complex scaffold was extended. Assessment of both cellular response and related gene expression demonstrated that the complexes did not initiate an immune response. However, Label-Free Quantification proteomic data indicated the larval response was associated with upregulation of key proteins such as Glutathione S-transferase, purine synthesis and glycolysis/gluconeogenesis (e.g. fructose-bisphosphate aldolase and glyceraldehyde-3-phosphate). Both Cu-Phen and Cu-DPQ-Phen elicited a similar in-vivo response in contrast to Cu-DPPZ-Phen, which displayed a substantial increase in nitrogen detoxification proteins and proteins with calcium binding sites. Overall, the response of G. mellonella larvae exposure to the complex series is dominated by detoxification and metabolic proteome response mechanisms.